Molecular characterization of the symbionts associated with marine nematodes of the genus Robbea^‡

Abstract

Marine nematodes that carry sulfur-oxidizing bacteria on their cuticle (Stilbonematinae, Desmodoridae) migrate between oxidized and reduced sand layers thereby supplying their symbionts with oxygen and sulfide. These symbionts, in turn, constitute the worms' major food source. Due to the accessibility, abundance and relative simplicity of this association, stilbonematids may be useful to understand symbiosis establishment. Nevertheless, only the symbiont of Laxus oneistus has been found to constitute one single phylotype within the Gammaproteobacteria. Here, we characterized the symbionts of three yet undescribed nematodes that were morphologically identified as members of the genus Robbea. They were collected at the island of Corsica, the Cayman Islands and the Belize Barrier Reef. The surface of these worms is covered by a single layer of morphologically undistinguishable bacteria. 18S rDNA-based phylogenetic analysis showed that all three species belong to the Stilbonematinae, although they do not form a distinct cluster within that subfamily. 16S rDNA-based analysis of the symbionts placed them interspersed in the cluster comprising the sulfur-oxidizing symbionts of L. oneistus and of marine gutless oligochaetes. Finally, the presence and phylogeny of the aprA gene indicated that the symbionts of all three nematodes can use reduced sulfur compounds as an energy source.

First detection of thiotrophic symbiont phylotypes in the pelagic marine environment

“…Nevertheless, putative free-living forms of these three thiotrophic symbionts have only been found in the vicinity of their hosts (Gros et al, 2003;Aida et al, 2008;Harmer et al, 2008). The lack of evidence for coevolution between the stilbonematid nematodes and their symbionts (Bayer et al, 2009) and the intermingled phylogeny of oligochaete and nematode symbionts within the MONTS cluster indicate that in the course of evolution, multiple recruitment events between hosts and associated bacteria occurred. The pelagic members of the MONTS cluster might represent the pool of environmental bacteria from which symbionts may be -or may have beenrecruited by the hosts.…”

“…Only one bacterial morphotype and one 16S rRNA gene phylotype were found to be associated to each given Robbea species. The presence and phylogeny of the aprA gene indicates that the bacterial symbionts may use reduced sulfur compounds as an energy source (Bayer et al, 2009).…”

“…Metagenomic analysis allowed the identification of Gamma 1 symbiont genes encoding for enzymes involved in CO 2 fixation via the Calvin cycle, the oxidation of reduced sulfur compounds (such as adenosine 5 0 -phosphosulfate reductase, AprA), and sulfur-storage, supporting its chemoautotrophic, sulfur-oxidizing nature (Woyke et al, 2006). In 16S rRNA gene-based phylogenetic trees, oligochaete Gamma 1 symbionts form a distinct cluster within the Gammaproteobacteria together with marine nematode symbionts (Musat et al, 2007;Dubilier et al, 2008;Bayer et al, 2009;Bulgheresi et al, 2011). We will refer to this as the marine oligochaete and nematode thiotrophic symbionts (MONTS) cluster.…”

All the microbiology nematodes can teach us

“…Ecological studies performed in the 90s suggest that stilbonematids trophically depend on their ectosymbionts, and these, in turn, profit from nematode migrations through the sulfide gradient in the marine sediment (Ott et al 1991). All the molecularly identified ectosymbionts belong indeed to the marine oligochaete and nematode thiotrophic symbiont (MONTS) cluster, which comprises 16S rRNA-gene sequences retrieved from gammaproteobacterial sulfur oxidizers associated with these invertebrates, as well as sequences of environmental origin (Polz et al 1994;Bayer et al 2009;Bulgheresi et al 2011;Heindl et al 2011;Pende et al 2014). The closest cultivable relatives of MONTS members are free-living purple sulfur bacteria (Chromatiaceae).…”

“…Beside the 16S rRNA-gene-based phylogenetic placement, the autotrophy of the symbionts is supported by uptake of 14C bicarbonate (Schiemer, Novak and Ott 1990) and by the presence of RuBisCo enzymatic activity (Polz et al 1992). As for the symbiont sulfuroxidation capability, it is supported by the ATP sulfurylase and sulfite oxidase enzymatic activities, by the presence of elemental sulfur in symbiotic but not in aposymbiotic L. oneistus (Polz et al 1992), and by the cloning of the symbiont aprA gene, encoding the alpha subunit of adenosine-5-phosphosulfate reductase (Bayer et al 2009). Moreover, metabolic studies suggest respiratory reduction of nitrate and nitrite (Hentschel et al 1999).…”